A conserved biological regulatory mechanism that controls eukaryotic gene expression has been found to utilize double-stranded RNA (dsRNA). This silencing response is known as RNAi. Perfectly paired dsRNA is processed into short interfering RNA (siRNA), which triggers silencing of genes related by sequence to the siRNA. Certain dsRNA that is transcribed from animal genomes is processed into microRNA (miRNA), which represses the expression of protein-coding genes. The long-term goals of our research are to understand the molecular mechanisms of siRNA and miRNA action, and to understand what aspects of cell and organismal biology are controlled by their activities. We are addressing these issues by studying Drosophila melanogaster. We conducted a screen for mutations that perturb the siRNA pathway. In addition to identifying essential processing and effector complex components, we identified several regulatory factors of the pathway: a Drosophila homolog of a F-box ubiquitin ligase FBXO11, and the Drosophila homolog to human HPS4, which is a protein associated with recycling endosomes. To systematically identify genes important for the miRNA pathway, we have also been conducting a screen for mutations that perturb the miRNA pathway in the compound eye. These studies have thus far led to the identification of four genetic loci that are candidates to encode factors acting at various steps in the miRNA pathway. The goal of the proposed research is to further decipher the mechanism of RNAi by: 1) completing the mutagenesis screens for genes required for the miRNA or siRNA pathways, and cloning these genes, 2) characterizing the roles of dFBXO11 and ubiquitination in controlling the siRNA pathway, 3) determining the functions of dHPS4 and other repressors of the siRNA pathway. The ability of RNAi to regulate viral, transposon, and organismal gene expression suggests that an understanding of RNAi will reveal basic control mechanisms that influence many aspects of biology. Moreover, RNAi has been used experimentally to probe gene function and holds great promise as a therapeutic model, making its basic understanding an important biomedical research goal.